In mice, the basic Helix-loop-helix transcription factor Neurogenin 3 (NEUROG3) is essential for development of the endocrine lineage in both the pancreas and the intestines. Humans born with NEUROG3 mutations suffer from congenital loss of enteroendocrine cells (enteric anendocrinosis) 1 but surprisingly had normal C- peptide levels and tolerate intravenous infusion of glucose, suggesting that NEUROG3 function is not required for pancreatic beta-cell development in humans. Our new preliminary data supports this conclusion. This application with explore the cause for the apparent lack of a pancreatic endocrine phenotype in humans. We will determine if endocrine pancreas development occurred due to a partial loss of NEUROG3 activity, or alternatively NEUROG3 function is not required development of human endocrine pancreas. Identifying the role of NEUROG3 is critical since it is considered a therapeutic linchpin for generating pancreatic endocrine cells from human ES cells or via neogenesis from adult cell types. Given the rarity of tissues from patients harboring NEUROG3 mutations, it has been impossible to mechanistically investigate the basis for this difference in intestinal versus pancreatic endocrine cell development. To enable such a developmental study, we have established a culture system whereby human embryonic and induced pluripotent stem cells (collectively called PSCs) are efficiently differentiated into eithe pancreatic or intestinal tissue in vitro. Our novel method for PSC-derived intestinal tissue generates an epithelium with all of the major functioning cell types of the gut, including enteroendocrine cells. Moreover we have been able to knock down NEUROG3 in the intestinal epithelium resulting in nearly complete loss of human enteroendocrine cells. We will use development of pancreatic and intestinal endocrine cells from human PSCs as a model to identify NEUROG3-dependent and independent pathways that regulate human endocrine pancreas development. We will first determine the impact of NEUROG3 levels on pancreatic and intestinal endocrine development from human PSCs in vitro. We will identify the molecular basis for the differential requirement for NEUROG3 between the gut and pancreas by looking at the effects of NEUROG3 levels on known targets and novel pathways. Lastly we will investigate the impact of NEUROG3 levels on pancreatic and intestinal cell function in vitro and in vivo. Together, our proposed aims constitute an unprecedented functional study of NEUROG3 during human pancreas and intestinal development. Our studies will unambiguously determine if NEUROG3 is required for the development of pancreatic versus intestinal endocrine cells and if it is not required, identify the molecular pathways that compensate for absence of NEUROG3. PUBLIC HEALTH RELEVANCE: Glucose homeostasis involves the complex activity of a number of hormones produced by the endocrine cells of the pancreas and intestine. Communication between the intestinal and pancreatic endocrine cells is central to glucose homeostasis. Derangement of this system can lead to loss of beta-cell mass and/or function and result in Diabetes Mellitus. Due to the central role of pancreatic and intestinal endocrine cells i glucose homeostasis, understanding the origin and function of these cells has been the focus for current and future diabetic therapies. For example, pharmacologic manipulation of the incretin response has been an effective approach in improving the function of pancreatic beta-cells in patients with Type 2 diabetes. The pancreatic hormone insulin has been used for decades to maintain glucose homeostasis in patients with Type 1 diabetes, and production of beta-cells from stem cells continues to be a focus for many researchers as a future therapy. Studies in mice suggest that NEUROG3 is a therapeutic linchpin to generate pancreatic endocrine cells from ES cells or via neogenesis from adult cell types. This proposal will unambiguously determine if NEUROG3 is, or is not, required for both beta-cell and intestinal enteroendocrine development in HUMANS. In either case, these studies will have broad impact in the diabetes research community by determining the relevance of NEUROG3 and by identifying compensatory, alternative pathways involved in human beta-cell development.